Arthropod-borne virus (arbovirus) diseases remain a significant burden on global public health. Maintenance of arboviruses in nature requires a biological transmission cycle that involves alternating virus replication in a susceptible vertebrate host and a blood feeding arthropod. While the vertebrate infection is acute and often associated with disease, continual transmission of virus depends on the establishment of a persistent, non-pathogenic infection in the arthropod. Both the virus and arthropod host clearly benefit from this non-pathogenic infection. Indeed, current models of vectorial capacity predict that transmission rates will be highest when arboviral infection has no effect on mosquito survival. The existence of endogenous mechanisms that quantitatively limit virus yields, termed modulation, has been advanced as an explanation for the apparent resistance of mosquito cells to the potential cytopathic effects of arboviruses. However, this has never been demonstrated experimentally. Nor have previous observations of modulation, made by arbovirologists over a period of more than 30 years, been directly linked to a specific immune mechanism. Our studies have established a link between arbovirus modulation, and recent evidence indicating that RNA interference (RNAi) functions as an innate anti-viral immune response in arthropods. Accomplishing the specific aims outlined here is designed to provide a comprehensive assessment of intrinsic and extrinsic factors influencing the relationship between RNAi-based modulation and the establishment of productive arbovirus infections in the mosquito. The specific goals of the proposed research are to 1) characterize the mechanism by which alphaviruses limit immune modulation in natural vector hosts, 2) determine the precise effects of temperature on RNAi and RNAi-based modulation of alphaviruses, and 3) determine the role of an RNAi-based immune response in modulating flavivirus infections in natural vector hosts. We believe that understanding how arboviruses interact with RNAi- based modulation will address glaring gaps in our knowledge of mosquito vector competence, and may ultimately form the basis of RNAi-based transgenic and field diagnostic approaches for disease control. PUBLIC HEALTH RELEVANCE: Arthropod-borne virus (arbovirus) diseases such as yellow fever remain a significant burden on global public health. This proposal deals directly with select agent and biosafety level 3 viruses which cause encephalitis or hemorrhagic fever. We aim to describe mechanisms used by these pathogens to successfully infect mosquito vectors. This knowledge is vital to understanding and predicting how arboviral epidemics are initiated, spread, and maintained, and may ultimately form the basis of RNAi- based transgenic and field diagnostic approaches for disease control.